Homotropic allosteric regulation in monomeric mammalian glucokinase.

Abstract

Glucokinase catalyzes the ATP-dependent phosphorylation of glucose, a chemical transformation that represents the rate-limiting step of glycolytic metabolism in the liver and pancreas. Glucokinase is a central regulator of glucose homeostasis as evidenced by its association with two disease states, maturity onset diabetes of the young (MODY) and persistent hyperinsulinemia of infancy (PHHI). Mammalian glucokinase is subject to homotropic allosteric regulation by glucose-the steady-state velocity of glucose-6-phosphate production is not hyperbolic, but instead displays a sigmoidal response to increasing glucose concentrations. The positive cooperativity displayed by glucokinase is intriguing since the enzyme functions as a monomer under physiological conditions and contains only a single binding site for glucose. Despite the existence of several models of kinetic cooperativity in monomeric enzymes, a consensus has yet to be reached regarding the mechanism of allosteric regulation in glucokinase. Experimental evidence collected over the last 45 years by a number of investigators supports a link between cooperativity and slow conformational reorganizations of the glucokinase scaffold. In this review, we summarize advances in our understanding of glucokinase allosteric regulation resulting from recent X-ray crystallographic, pre-equilibrium kinetic and high-resolution nuclear magnetic resonance investigations. We conclude with a brief discussion of unanswered questions regarding the mechanistic basis of kinetic cooperativity in mammalian glucokinase.

Steady-state kinetic assays of the wild-type human glucokinase fitted to the Hill equation. The data are plotted as a function of glucose concentration to indicate the positive cooperativity in the rate of glucose-6-phosphate formation as a function of glucose concentration. The sigmoidal response is most noticeable at low glucose concentrations (inset).

Conformational changes experienced by the C-terminal α-13 helix upon glucose binding. (A) The α-13 helix (red) is solvent exposed and released from the connecting loop (yellow) in the unliganded state. (B) In the presence of glucose and an allosteric activator (not shown), the α-13 helix is constrained by the connecting loop. Experiments indicate that fusion of additional residues at the C-terminus has no effect upon steady-state kinetic properties, an observation that is difficult to reconcile with this structural data. Image was generated using PDB entry 1V4T, 1V4S [] and PyMol 1.1 [].

Kinetic mechanisms used to describe glucose association with mammalian glucokinase resulting from various transient state binding studies. (A) Kinetic scheme proposed by Neet and coworkers involving the fast formation of a binary enzyme-glucose complex, followed by a slow reversible isomerization []. (B) Kinetic model for glucose binding to human pancreatic glucoskinase, as proposed by Heredia et al., based on KIMSIM simulations []. (C) The Kim model, which includes a preexisting equilibrium to describe the transient-state glucose binding traces []. The values are obtained from the simulated dependence of kobs with increase glucose concentrations. (D) The Antoine et al. model, which includes a preexisting equilibrium to describe the transient state glucose binding traces. The kinetic values shown are obtained from the simulated dependence of kobs3 with increasing glucose concentration []. (E) The Larion model, which resulted from global fitting of rate and amplitude data of glucose binding traces at high enzyme concentrations. The calculated KD for the high-affinity state, E, is 782 µM. The calculated KD for the low-affinity state, E*, is 114 mM [].